Project Summary: First synthesized in the 1950s, benzodiazepines (BZDs) are widely prescribed drugs that exert their anxiolytic, muscle relaxant, sedative, and anticonvulsant actions by binding to GABA-A receptors (GABAARs), the main inhibitory ligand-gated ion channel in the brain. Since their synthesis, many scientists have theorized that there exists an endogenous BZD, or endozepine, in the brain. In 1983, a candidate polypeptide that displaced BZDs in a GABAAR binding assay was isolated from brain homogenates . However, proof that this 1 peptide called diazepam-binding inhibitor (DBI) had functional effects in the brain was lacking. Recently, using DBI knock-down experiments it was demonstrated in the subventricular zone of the lateral ventricles that DBI and one of its peptide fragments, ODN, inhibit GABA-induced currents and promote neurogenesis , indicating that DBI and ODN work as negative allosteric modulators of GABAARs in vivo. 2 However, in the thalamic reticular nucleus (a nucleus that plays an important role in controlling epileptic seizures), DBI and/or a DBI-derived peptide was found to enhance GABAAR currents and suppress epileptic activity . Thus, depending on the brain region, DBI can potentiate or inhibit GABAAR activity. Little, however, is 3 known about how DBI exerts these actions. How do products from a single protein elicit positive versus negative effects on GABAAR currents? Do DBI- derived peptides such as ODN elicit the same functional effects as DBI? Do different cell types make and process distinct DBI peptide(s)? Is DBI modified post-translationally (e.g. phosphorylated, acylated) and can these modifications alter DBI's physiological effects? The proposed experiments will address these fundamental questions and will 1) determine if specific GABAAR subunit combinations mediate the positive and negative actions of DBI and ODN; 2) determine if astrocytes and neurons release different DBI peptides and/or post-translationally process DBI differently, which impact its functional effects and 3) determine if astrocytes and neurons express different DBI transcript variants. Successful completion of these experiments will validate DBI's role as an endogenous BZD and will lay the foundation for understanding how DBI regulates GABA- mediated inhibition in the brain.